Medicinal chemistry perspective of pyrido[2,3-d]pyrimidines as anticancer agents

Cancer is a major cause of deaths across the globe due to chemoresistance and lack of selective chemotherapy. Pyrido[2,3-d]pyrimidine is an emerging scaffold in medicinal chemistry having a broad spectrum of activities, including antitumor, antibacterial, CNS depressive, anticonvulsant, and antipyretic activities. In this study, we have covered different cancer targets, including tyrosine kinase, extracellular regulated protein kinases – ABL kinase, phosphatidylinositol-3 kinase, mammalian target of rapamycin, p38 mitogen-activated protein kinases, BCR-ABL, dihydrofolate reductase, cyclin-dependent kinase, phosphodiesterase, KRAS and fibroblast growth factor receptors, their signaling pathways, mechanism of action and structure–activity relationship of pyrido[2,3-d]pyrimidine derivatives as inhibitors of the above-mentioned targets. This review will represent the complete medicinal and pharmacological profile of pyrido[2,3-d]pyrimidines as anticancer agents, and will help scientists to design new selective, effective and safe anticancer agents.


Introduction
Cancer is the explosive growth of abnormal cells that typically grow beyond their original boundaries, invade surrounding areas, spread to other organs, and result in metastasis, which is one of the main causes of cancer-related death,and second most common cause of death across the globe. Around 10.0 million cancer-related fatalities (9.9 million excluding nonmelanoma skin cancer) and 19.3 million new cancer cases (18.1 million excluding nonmelanoma skin cancer) were estimated in 2020 across the globe. The most lethal malignancies include lung (1.8 million), colorectal (935 000), liver (830 000), stomach (769 000), and breast cancer. The most frequent cancers worldwide are lung (2.2 million), breast (2.09 million), colorectal (1.9 million), prostate (1.28 million), skin (1 million), and stomach (1 million) (627 000). 1 Cancer is the Latin term meaning crab. Because of the crab-like tenacity, a malignant tumor oen appears to grab the tissues it invades, and the ancients used the term to indicate a malignancy. 2 Cancer cells (dened by their uncontrollable growth and invasion of other tissues) do not show the same growth average as a normal heathly cell. Normal cells multiply and expand, controlled to produce additional cells as required to maintain a healthy body. Cells die as they grow old or are damaged and are replaced by new cells. Cancer cells, rather than dying, continue to proliferate and generate new, aberrant cells. Normal cells cannot invade (grow into) other tissues. The other cells can combine to form a mass of tissues known as a tumor. Not all tumors are cancerous; they might be benign (not cancerous) or malignant (cancerous). 3 The worldwide burden of cancer is anticipated to rise signicantly due to population growth, age, and increase in risk factors like smoking, inactivity, poor food, and infections that cause cancer. It is well established that cancer diagnosis and treatment have substantial and long-term consequences on both patient and caregiver physical, emotional, and spiritual well-being. 3,4 2 Synthetic strategies of pyrido [2,3-

Tyrosine kinase
Tyrosine kinases are essential components of the signaling cascade that controls cell division, metabolism, migration, and the death of cells. Tyrosine kinases use ATP to catalyze the phosphorylation of particular tyrosine residues in their protein targets. 28 Tyrosine kinases play an essential role in several stages of the emergence and spread of cancer. Tyrosine kinase signal transduction typically regulates excessive proliferation or increases apoptotic sensitivity. These signaling pathways are frequently genetically or epigenetically changed in cancer cells to provide specic benets to cancer cells. As a result, it is no surprise that abnormally increased signaling from tyrosine kinases gives these enzymes a dominant oncoprotein status, resulting in signaling network failure. 29 3.1.1 Biological pathway and inhibition of tyrosine kinase. Receptor tyrosine kinase (RTKs) are usually activated by ligands that target specic receptors. Growth factor ligands bind to RTK's extracellular regions, and the receptor activation is carried out by ligand-induced dimerization or oligomerization. 30 Ligand attached to the extracellular domain stabilizes the generation of active dimers, which activates protein tyrosine kinases. The concentration of RTKs is increased by receptor oligomerization, enabling effective transphosphorylation of tyrosine residues inside the activation loop of the catalytic domain. For most RTKs, the resulting conformational alterations allow trans-autophosphorylation of each TKD and the release of cis-autoinhibition (Fig. 10). 31,32 Genetic amplication is the principal mechanism that leads to the overexpression of RTKs and transcription enhancement. 33,34 SH2 domain proteins interact with phosphorylated receptors and become phosphorylated as well. The growth factor Ras/Raf/mitogen-activated protein map phosphorylated cascade then connects with the substance of the g-protein system, allowing the cell to migrate and express itself. 30, 35 3.1.2 Mechanism of action of tyrosine kinase inhibitors. Several amino acids of substrate enzymes are phosphorylated by tyrosine kinases, leading to different changes in the downstream cellular biology and signaling. TKs can alter cell growth, migration, differentiation, apoptosis, and death by triggering downstream signal transduction. Constitutive activation or inhibition, whether caused by mutations or other methods, can result in dysregulated signal cascades, which can lead to cancer and other diseases. 35,36 The tyrosine kinase inhibitors inhibit this dysregulated signal cascade and block transcription in both genetic amplication and mutation, leading to the disease-causing signals. 36 The compounds 47-51 showed excellent activity against various kinases, including TKs, PI3K, and CDK4/6. In molecule 47, the presence of dichloro and tertiary amine increased the activity. In molecule 48, piperazine with amine linkage and acetyl group at pyridine ring increased the activity, thiophene, phenyl with uorine atom were responsible for activity in 49, presence of methoxy and morpholine were necessary for activity in molecule 50, also other triazole rings in 51 enhanced the activity (Fig. 11). [37][38][39][40] In 2018, Elzahabi et al. successfully synthesized substituted pyrido [2,3-d]pyrimidines and tested their ability to inhibit the growth of ve cancer cell lines. Pyrido [2,3-d]pyrimidines, were found to have signicant inhibitory effects against a variety of kinases, including TKs, PI3K, and CDK4/6. 9 3.1.3 Structure activity relationship of tyrosine kinase inhibitors. According to the structure-activity relationships of the screened products, presence of carbonyl at C-2 of pyrido [2,3d]pyrimidine derivatives (52)(53)(54)(55)(56) provided the maximum anticancer activity. Particularly 52 and 53, which had (4-CH 3phenyl) and (4-chlorophenyl) at C-5 and C-7, respectively, shared the same pyridopyrimidine scaffold. Compound 53 containing 3-methyl-5-oxopyrazolyl moiety of showed strong anticancer efficacy against prostate, colon, and liver malignancies. The anticancer action was switched from anti-colon to anti-prostate cancer when the methylpyrazolone moiety in 53 was replaced with aminopyrazolone in 54 ( Fig. 12 and 13).
In the same way, adding a steric bulky group to 56 and 57 reduced its anticancer efficacy. Converting the thioxo group in 58 to the hydrazide moiety in 59 signicantly enhanced the antihepatic cancer activity. The electronic component was able to have a positive impact on anticancer activity due to the hydrophilic electron-rich nature of the hydrazide molecule. 9 A novel class of substituted pyrido [2,3-d]pyrimidinones was investigated for their in vitro anticancer efficacy. Compounds 52 and 55 were more effective (IC 50 = 0.3 mM) than doxorubicin in the HepG-2 cell line. In addition, compound 59 was approximately equivalent to doxorubicin (IC 50 = 0.6 mM). Compounds 60 and 52 were having higher activity than doxorubicin (IC 50 = 5.47, 6.6, and 6.8 mM, respectively) in a cellular screening on PC-3. The activity of 60, 52, and 53 was two-fold as that of doxorubicin in the HCT-116 cell line (IC 50 of 6.9, 7, 5.9 mM, and 12.8 mM, respectively). Synthesized compounds showed signicant growth inhibitory effects in HepG-2, HCT-116 as well as PC-3 cell lines with reference to doxorubicin, but on MCF-7 and A-549 cancer cells showed comparetively low activity. PDGFR b, EGFR, and CDK4/Cyclin D1 kinases were all inhibited by the potent anticancer molecule 52 (Fig. 13). 9 Pyrido[2,3-d]pyrimidine is a vital core known to play a role in many active compounds with diverse activities, particularly chemotherapeutic, which may be capable of inhibiting cyclin-dependent kinase (CDK). For example, The US Food and Drug Administration has authorized the CDK4/6 inhibitor palbociclib (61), which is used to treat breast cancer. In addition, 2,4-    (64) showed greater efficacy against both cell lines, causing cell cycle arrest by increasing the cell cycle inhibitor p21 and cancer cell death by activating caspase-3 (Fig. 14). 41 Another study discovered a highly potent tyrosine kinase inhibitor (65) having IC 50  pyrimidine analogs. 44,45 In the Colo-205 as well as U87MG cell lines, CDK4/6 was found to be effectively suppressed by compounds 70 and 71. The equipotent of palbociclib and abemaciclib, compounds 70 and 71 signicantly inhibited CDK4/ D3 (IC 50 = 0.8 nM and 2.7 nM, respectively) and CDK6/D3 (IC 50 = 2.0 nM and 4.8 nM, respectively). The acetyl and isopropyl moieties at C 6 and C 8 positions (compound 70) represented 2.5 more possible biological activities for CDK4/cyclinD3 than CDK6/cyclinD3. 45 Changes to the functional group tertbutyl at the C 8 -substituent (compound 71) did not affect CDK4/6 targeting bioactivity. Compounds 70 and 71 showed favorable antiproliferative effects, remarkable metabolic properties, useful pharmacokinetic features, and signicant tumour growth inhibitions with manageable toxicity in Colo-205 and U87MG xenogra models (Fig. 15). 45 3.2 Extracellular signal-regulated kinase and Phospatidylinositol-3 kinasea 3.2.1 Extracellular regulated protein kinases (ERK). A serine/threonine protein kinase known as ERK1/2 (extracellular signal-regulated kinase) is frequently found in eukaryotic cells. It belongs to the family of MAPK (mitogen-activated protein kinases). The transfer of signals from surface receptors to the nucleus is signicantly aided by ERK1/2. Apoptosis, cell proliferation, differentiation, and other activities are regulated by activated ERK1/2, which phosphorylates substrates in the cytoplasm or nucleus and causes the production or activation of particular proteins. 46 3.2.2 Phospatidylinositol-3 kinase. Many cancers, including breast, gastric, ovarian, colorectal, prostate, glioma, and endometrial cancers, have altered or amplied the activity of the critical intracellular signalling system known as PI3K (phospatidylinositol-3 kinase, or PI3K). PI3K signalling is a viable therapeutic target since it is crucial for cancer cell survival, angiogenesis, and metastasis. Clinical trials employing PI3K inhibitors are continuing and have already been completed to identify effective PI3K inhibitors that could overcome resistance to existing treatments. 47 Critical physiological and pathological processes like metabolism, cell development, proliferation, angiogenesis, and metastasis are inuenced by PI3K/AKT signalling and contribute to the progression of tumours. 48,49 PI3K proteins are categorized into three major classes based on the structural characteristics of the proteins as well as the peculiarities of the substrates (I, II, and III). Class, I PI3Ks are classied into subtypes A and B based on how they are regulated. Class IA PI3Ks combine their regulatory (p85, p85, p55, p55, and p50) and catalytic (p110, p110, p110) subunits to form dimers. 50 3.2.3 ERK2 and PI3Ka. The combined inhibition of the MAPK and PI3K signalling pathways has been identied as a viable cancer therapy that effectively overcomes the drug resistance of MAPK signalling pathway inhibitors. 51 Zhang et al. reported the scaffold-hopping synthesis of ERK/ PI3K dual inhibitors by replacing 1H-pyrazolo [3,4- 51 Compounds 73-75 were foundd to be dual inhibitors of ERK2 and PI3K-a. Compound 74, a potent and very effective ERK and PI3K dual inhibitor, was discovered during preliminary SAR study. Compound 74 had modest ERK and PI3K inhibitory and anti-proliferative properties. Although 74 had only acceptable pharmacokinetic characteristics in SD rats, with a moderate half-life (t 1/2 = 2.32 h) aer intravenous treatment, it displayed signicant anticancer activity in vivo in an HCT-116 xenogra model without eliciting obvious adverse consequences. 51
In contrast, the 1-phenylpiperazine, a methoxy group at the fourth position, decreased absorbance, rendering molecule 92 more efficient than molecule 91. This demonstrated that enhanced activity may occur when a piperazine moiety is modied at place four with a phenyl ring modied with an electron-donating compound, but not when an electronwithdrawing group or even with an unsubstituted group. It has been demonstrated that compound 93's methoxy group on fourth place of the Schiff base aromatic ring was more active than compound 94's nitro group. The presence of an electrondonating group on an aromatic nucleus is associated with increased anticancer action in part. 62

BCR-ABL kinase
BCR-ABL is a mutation that results from the interaction of two genes, BCR and ABL. It is also known as a fusion gene. The BCR and ABL are present on chromosomes 22 and 9, respectively. When portions of the BCR and ABL genes break off and swap locations, the BCR-ABL mutation occurs. BCR-ABL genes were found in patients with certain types of leukemia and also detected in almost all patients with chronic myeloid leukemia (CML). 63 Loss of auto-inhibition and constitutional activation of ABL1 may occur when the mutant BCR-ABL1 protein arises, with subsequent effects on signalling pathways related to cell cycle and apoptosis, such as the RAS/RAF/MEK/ERK pathway, the JAK2/STAT pathway, and the PI3K/AKT/mTOR pathway. Finally, it promotes the malignant transformation of hematopoietic cells. 64 (95) inhibited Bcr-abl activity in cells. In these cells, IC 50 for the suppression of Bcr-abl autophosphorylation was 1 nm as compared with 100 nm for STI571. The biological activity and potency of PD166326 (95) was tested in K562 cells in cell growth assays. With an IC 50 of 0.3 nm, compound 95 suppressed K562 cell growth. Additional hematopoetic and epithelial cell lines were only inhibited at two to three log higher doses with IC 50 s in the 0.8-2 nm range, indicating that PD166326's powerful biological activity was highly selective for Bcr-abl-driven cells. 68 Bubnoff et al. tested 13 different pyridopyrimidine derivatives against wild-type as well as mutant BCR-ABL. All the compounds decreased the Bcr-Abl dependent phenotype more effectively than imatinib and inhibited Bcr-Abl kinase activity (Table 1). PD166326 (95) and SKI DV-M016 (96) were the most active compounds. Surprisingly, these drugs repressed Bcr-Abl H396P, a mutant with an activation loop, just as well as wild-type Bcr-Abl (Fig. 19). 65 3.4.1 Mechanism of action of BCR-ABL inhibitors:-. BCR-ABL inhibitors compete with adenosine triphosphate (ATP) for the same binding site inside the BCR-ABL protein's kinase domain. When ATP is present in the binding site, BCR-ABL can catalyze the phosphorylation of tyrosine residues on substrate proteins. These BCR-ABL inhibitors (Imatinib mesylate) have a structure similar to ATP and can bind to the BCR-ATP-binding ABL's region. Because BCR-ABL inhibitors lack the necessary phosphate groups given by ATP, phosphorylation of tyrosine residues on substrate proteins is impossible. Unphosphorylated tyrosine residues in substrate proteins prevent them from adopting the required conformation for binding to effector molecules. As a result, downstream signal transduction pathways important for CML leukemogenesis are blocked (Fig. 20). 69 3.5 p38 mitogen-activated protein kinases p38 Mitogen-activated protein kinases (p38 MAPKs) are prolinedirected serine/threonine kinases that regulate pro-inammatory signalling networks and are involved in the production of cytokines such as tumour necrosis factor-a (TNFa) and interleukin-1b (IL-1b). 70,71 Four isoforms of p38 MAPK have been found, namely a, b, g, and d, with p38a MAPK being the earliest identied, best described, and most signicant isoform involved in the inammatory response. 72 The N-and Cterminal domains of p38a MAPK form the walls of a deep cle in which the ATP cofactor is allocated in a pocket known as the ATP binding site. 73 The adenine ring of ATP formed hydrogen bonds with His107 as well as Met109 residues of the p38a MAPK backbone. These two residues are found in the hinge region (residues 106-110), which connects the N and C-lobes, whereas the adenine binding region and ribose pocket, respectively, are located in the adenine binding region and ribose pocket, respectively. 74 The dual interaction of ATP with the hinge region aligns the two protein lobes to produce a pocket large enough to hold the ATP triphosphate group; this pocket is known as the phosphate binding area and contains highly conserved kinase residues like Glu71 and Asp168. 75 Three more hydrophobic areas have been discovered adjacent to the ATP binding site but are not directly involved in ATP binding (Fig. 6). 76 The rst section is known as hydrophobic region I (HR-I), and it is   (Fig. 21). 77 Because similar kinases have a bulkier residue at the same position, the tiny size of the gatekeeper residue Thr106 allows access to this pocket, which is essential for kinase inhibitor selectivity. The hydrophobic region II (HR-II), a solvent-exposed hydrophobic area dened mainly through residues Val30, Ile108, Gly110, Ala111, and Asp112, is the second section. 78 The allosteric site is located next to HR-I and is only accessible when the kinase adopts the catalytically inactive conformation (DFG-out), in which the highly conserved Asp168-Phe169-Gly170 (DFG) motif is ipped "out" in comparison to its active state conformation (DFG-in). 79 3.5.1 Structure-activity relationship of P38a inhibitors. The 6th-substituent substantially inuences inhibitors' pankinase selectivity. In terms of potency, this position is also critical. 6-Aryl substituents, such as o-chlorophenyl in compound 98, are generally potent compared to 99 (Table 2). Phenoxy substituents were chosen because of their high activities and intriguing prole of kinase selectivity. SAR revealed that the phenoxy group found having tiny ortho and para substituents are preferred over no and meta substitutions. The ortho and para diuoro substitution improved the side chain's metabolic stability. At the 8th-position of the scaffold, many different side chains are allowed, reinforcing the idea that this variable points towards solvent. The kinase selectivity of 2alkylamino side chains was higher than that of 2-anilino side  chains. The molecule's potency was increased by substituting the 4-tetrahydropyranyl group in the second position (Fig. 22). 22 p38 is one of many kinases implicated in a stress-response signaling pathway similar to but not identical to the MAP kinase cascade. Phosphorylation of threonine 180 and tyrosine 182 by stress signals activates p38. MAPKAP kinase-2 and MAPKAP kinase-3 have been found as downstream targets of p38, which phosphorylates heat shock protein (Fig. 23). 22 Dilmapimod (100, CAS 444606-18-2) is a p38 MAP-kinase inhibitor patented by SmithKline Beecham Corporation (GSK-    patented by Takeda Pharmaceutical Company in 2007. TAK-733 was tested against non-small cell lung cancer and progressed to Phase I before being recalled off the market in 2015 (Fig. 24). 16

Phosphatidylinositol 3 kinase and mTOR protein
The phosphatidylinositol (PI) second messengers PI(3)P, PI(3,4) P2, and PI (3,4,5)P3 are produced by phosphoinositide 3-kinase (PI3-KS) enzyme (PIP3). The Class I PI3-KC isoform, which consists of catalytic (p110C) and adapter (p85) subunits is the most common PI3-kinase isoform. By producing 3-phosphorylated phospholipids (PIPs), PI3-KS activates kinases with lipidbinding domains, such as pleckstrin homology (PH) regions, which function as second messengers. For example, Akt as well as phosphoinositide-dependent kinase-1, Akt is moved to the cellular membranes when it links to membrane PIPs, which bind with PDK1 and becomes activated. A tumour suppressor protein, PTEN, dephosphorylates PIP, inhibiting the activation of Akt. The Akt, PDK1, and PI3-Ks are crucial players in controlling numerous cellular functions, including motility, proliferation, survival, and apoptosis. They are also crucial for disease-related molecular pathways, such as cancer, diabetes, and immune-mediated inammation. Oncogenesis is linked to several components of the PI3K/Akt/PTEN pathway. 80 In 2007, Pzer received a patent for compound 102, inhibiting the mTOR protein and phosphatidylinositol 3 kinases. Due to undesirable effects, it's clinical trial to treat endometrial cancer was stopped in 2012. N68 has also been tested in clinical trials for early (Phase 2) and late breast cancer (Phase 1b). 16 Finally, Voxtalisib (CAS 934493-76-2) (103), a kinase inhibitor of PI3K and mammalian target of rapamycin in the PI3K/mTOR signaling cascade was patented by Exelis (XL-765) in 2006. In 2018, withdrawal of voxtalisib as a monotherapy and combo therapy for solid tumors (breast and ovarian cancer) took place due to serious side effects (Fig. 25). 16 In the discovery of anticancer therapies, the mammalian target of rapamycin (mTOR) is a critical target. 81 The growth factor/mitogenic activation of the phosphatidylinositol 3-kinase (PI3K/Akt) signalling pathway activates this key regulator of cell growth and proliferation, frequently dysregulate route responsible for diseases. Serine and theonine kinases are the 2 mTOR found in the mammalian body. It's about 289 kDa in size and belongs to the evolutionary family. The eukaryotic PI3K like kinase (PIKK) family of proteins is highly conserved. [82][83][84] 3.6.1 Mammalian target of rapamycin pathway. The rapamycin and its analogues (RAD001, CCI-779, AP23573) bind to the FKBP12/rapamycin complex binding domain (FRB) and suppress signaling downstream substrates p70S6K and 4E-BP1. 84-86 LY294002 and wortmannin, potent but non-specic PI3K inhibitors, have also been demonstrated to block the kinase activity of mTOR by targeting the protein's catalytic domain. 87 According to the Brunn et al. and Edinger et al. mTOR is divided into two complexes: mTORC1, a rapamycin-sensitive complex that signals to p70S6K and 4E-BP1, and mTORC2, a rapamycin-insensitive complex that signals to Akt. 87,88 Therefore, direct targeting of the mTOR kinase domain would suppress signalling via both mTORC1 and mTORC2 and such a drug would have a distinct pharmacological spectrum than rapamycin (Fig. 26). 84 3.6.2 Structure-activity relationship of mTOR inhibitors. The dimethyl substitution at C 2 of the morpholino group balances the compound's, resulting in strong mTOR inhibitory activity ( Table 3). The presence of the morpholino group is required for mTOR inhibitory activity. The oxygen in this group mediates the H-bond interaction with the mTOR kinase. The potency is signicantly reduced due to the slight change in structure and increase in bulk. The combination of an electron donor at the para position with a hydrogen bond donor at the meta position in the phenyl ring exhibited a strong mTOR inhibitory effect (Fig. 27). 84 The KU-63794 (compound 108) indicated that mTOR selectivity has been achieved compared to other PIKK family members. Furthermore, it exhibited no signicant activity when tested on 200 kinases (non-PI3K related) at a concentration of 10 mM. U87MG glioblastoma cells were treated with the KU-63794 (108) for 2 h to see whether it inhibited both mTORC1 and mTORC2 complexes in the cellular environment. Using a western blot, the phosphorylation of the direct mTORC2 substrate Akt (Ser473) and the indirectly mTORC1 downstream signalling S6 ribosomal protein (Ser235/236) was determined. The IC 50 s were 0.10 and 0.15 mM for each of the endpoints.

Targeted inhibition of dihydrofolate reductase
NADPH (Nicotinamide adenine dinucleotide phosphate) serves as a cofactor for dihydrofolate reductase (DHFR), which transforms dihydrofolate (DHF) into tetrahydrofolate (THF). THF is necessary to form de novo purine and thymidylate (TMP) during cell growth. 89 Inhibiting DHFR activity causes cell death, 90 making DHFR an essential target enzyme in developing chemotherapy. 91 Antifolates are categorized into two types: traditional/Classical: Due to the inhibitor's hydrophilic characteristic and glutamate moiety (which prevents the inhibitors from diffusing into cells), an active transport mechanism is needed. However, in the non-classical form, substituting the hydrophilic glutamate moiety with lipophilic groups improves its passive diffusion into cells. 92,93 Dihydrofolate reductase (DHFR), specic kinases, such as the tyrosine-protein kinase transforming protein Abl or MAP kinases, and the biotin carboxylase are the most commonly described biological targets of pyrido[2,3-d]pyrimidine derivatives (Fig. 28). 18 Walaa  effects and were tested for enzymatic inhibition of the dihydrofolate reductase [compared to the reference medication methotrexate (MTX)] to explain the likely mechanism of action of the observed anticancer activity. 94 Molecule 115 had the highest inhibitory activity at IC 50 of 6.5 mM among all the tested molecules (IC 50 of MTX = 5.57 mM, Table 4).
3.7.1 Structure-activity relationship study of DHFR inhibitors. SAR study indicated that pyrido[2,3-d]pyrimidine scaffold was essential for activity. If the R was replaced with 2,5-dimethoxy substituents, it acted as PTX, and modication at NH position enhanced the activity. Both NH 2 groups were responsible for hydrogen bonding (Fig. 30).

Apoptosis inducer
3.8.1 Cyclin-dependent kinase (CDK). In cancer, the cellcycle regulatory mechanism, which is necessary for cellular division, is commonly disturbed, resulting in tumour growth. CDK4/6 inhibition inhibits cell proliferation and suppresses  95 Protein kinases, CDKs (cyclin-dependent kinases), are crucial for cellular processes. They participate in vital physiological processes like transcription and the cell cycle. Based on sequence similarities, the human genome contains 21 genes that encode CDKs, and ve genes that encode a more distant group of proteins have been identied as CDK like (CDKL) kinases. The current CDK nomenclature includes 11 classical CDKs (CDK1-11), two newly proposed family members (CDK12-13), and additional proteins whose names are based on the presence of a cyclin-binding element (PFTAIRE and PCTAIRE proteins) or simply on sequence similarity to the original CDKs, such as CDC2-like kinases (CDC2L) or Cell cyclerelated kinases (CCRK). 96 Apoptosis means allowing cells to die in a controlled manner. Apoptosis is a tightly controlled process in which cells self-destruct and eliminate undesired or diseased cells. The cell genome fractures, shrinks, and the cell portion disintegrate into smaller apoptotic entities during apoptosis. 97 Cancer cells can potentially resist apoptosis, resulting in uncontrolled cancer development. 98 The standard cellular mechanism called apoptosis keeps the ratio of healthy living cells to healthy dying cells in equilibrium. As a result, triggering apoptosis in cancer cells may be viewed as a cancer therapy strategy that does not harm healthy cells and prevents tumour resistance. As a result, nding novel drugs that target any phase of the cell cycle that leads to apoptosis might be a promising anticancer strategy. 99 3.8.2 Cyclin-dependent kinase (CDK4/6). Pyrido[2,3-d] pyrimidine is a critical component in various bioactive compounds, including anticancer medicines, attributed to cyclin-dependent kinase inhibition. 100,101 Palbociclib is a CDK4/  Apoptosis assay, 105 Caspase activation assay, 106 RT-PCR for (Bax, Bcl2,p53, and CDK4/6), and compound 116 showed excellent activity against Caspase-3 (Table 5). Furthermore, compounds 116 and 117 showed an excellent docking score and primary amino acid interaction with selected protein (  107 and the proper regulation of CDK activity is crucial for the ordered execution of the cell cycle. 108 CDK2/cyclin E expression abnormalities have been seen in colorectal, ovarian, breast, and prostate cancers. 109 CDK2 is involved in many stages of the cell cycle, including DNA repair, gene transcription, the G1-S transition, and G2 progression modulation. [110][111][112] 3.8.4 The function of CDK2 in the proliferation of cells: -. CDK2 is involved in cell cycle control, transcription activity, and tumor epigenetic alterations. Certain malignancies, such as breast cancer, 113 colo-rectal cancer, 114 glioblastoma, 115 and melanoma 116 may benet from CDK2 inhibition (Fig. 32).
Cyclin-dependent kinase promotes the progression of the G1 phase into the S phase by phosphorylation of the retinoblastoma protein (RB). The E2F family of transcription factors is activated by the hyperphosphorylation of the RB, which reduces its capacity to regulate growth. Growth repressive signals promote CDK inhibitors from the INK4 and CIP/KIP families, which block G1-S progression. Cyclin and dependent kinase complexes are activated by mitogenic signals. 117,118 Diaa et al.,   (118) 92.78 pyrimidines by introducing a variety of substituents at C-2, C-5, and C-7 of the pyridopyridine ring for a potent and selective inhibitors of CDK2 (competitive inhibitors of ATP binding site) and tested them for CDK2, EGFR inhibitory activities, and cell growth inhibitory activities. 10 Using the design of structural scaffolds addressing ATP interactions, they created pyrido [2,3d]pyrimidine scaffolds (CDK2 inhibitors) using the X-ray crystalline structure of CDK2 (PDB ID: 1HCK). The CDK2 inhibitory and anti-proliferative activity is due to the side chains at the 2nd and 7th positions. The CDK2, CDK4, EGFR, and antiproliferative activities of the Pyrido[2,3-d]pyrimidine derivatives were assessed against SNU638, A431, and HCT116, and the results showed that compounds 119 and 120 were more active than roscovitine with IC 50 values of 0.3 and 0.09 mM, respectively, which shows that compound 120 has the good antiproliferative activity. The compounds did not show any significant activity against the EGFR, which shows their selectivity against the CDK2 (Fig. 33). 10 3.8.5 Structure activity relationship of CDK2 inhibitors. SAR studies relealed that 7-amino group's lipophilic function (LF), such as the benzene, and sulfonamide group, signicantly increased the CDK2 inhibitory effect. Further, small (LF) with an electron donating group on position 2 of the pyrido[2,3-d] pyrimidine moiety was required for CDK2 inhibitory activity.

Phosphodiesterase inhibitors
Phosphodiesterase 3 is also known as the cyclic GMP-inhibited PDE family. PDE3A and PDE3B are the two genes in the family, each has numerous splice variants. Harrison et al., Macphee et al., Maurice and Haslam et al. were the rst to discover PDE3A as a signicant PDE isoform in bovine heart and platelets. 119 PDE3 enzymes play a role in modulation of cardiac and vascular smooth muscle contractility. PDE3 inhibitors were once examined for the treatment of heart failure. However, they are no longer being studied for that due to undesired arrhythmic side effects. The PDE3 inhibitor milrinone is licensed for intravenous usage in the treatment of heart failure. 120    aminophylline affect the proliferation of cancer cell lines. 122 HMG (Human malignant melanoma) has been found to have PDE3 activity, indicating that it might be a potential target for antineoplastic medications. However, the PDE3-specic inhibitors trequinsin and cilostamide did not affect HMG cell growth. 123 The expansion of colon cancer cells was associated with the upregulation of PDE3B mRNA as well as protein in HT-29 cells. Through the dose-dependent activation of PDE3B, cyclic phosphatidic acid (cPA) reduced Akt phosphorylation, lowering HT-29 cell proliferation. As a result, the cPA-PDE3B-cAMP pathway plays an essential role in colon cancer growth, and cPA could be exploited for colon cancer targeted therapy. 124 3.9.1 Mechanism of action of phosphodiesterase inhibitors. The cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) signalling systems are among the earliest signalling systems discovered, and are involved in a variety of physiological processes such as visual transduction, cell proliferation and differentiation, cell-cycle regulation, gene expression, inammation, apoptosis, and metabolic function. 125 Adenylyl-cyclase and guanylyl-cyclase produce cyclic nucleotides when G-protein-coupled receptors and molecules like natriuretic peptide or nitric oxide have been activated. cAMP then activates cAMP-dependent kinase (PKA) and other proteins, regulating biological processes like cell differentiation and proliferation. Proteins are phosphorylated when cGMP activates the cGMP dependent protein kinase (PKG), which is involved in physiological mechanisms like ion channel conductance as well as cell death. Phosphodiesterases 3 (PDEs) regulate cAMP and cGMP intracellular concentrations and their diverse biological consequences by catalyzing their hydrolysis. 122 PDEs can be expressed by every cell type, and their location is crucial in controlling cAMP or cGMP cellular functions. 126 Interference with the cAMP/Cgmp signalling pathway has been linked to tumour growth. Increased intracellular cAMP/cGMP may suppress tumour growth and hence be a protective mechanism against tumour progression. 3 PDE inhibitors that limit cAMP or cGMP hydrolysis have thus been identied as possible anticancer medicines (Fig. 34). 125 However, some cancers, such as adenocarcinoma, can stimulate cell proliferation when cAMP levels are elevated. 127,128 A cyclized pyridopyrimidone analog (122) has an IC 50 as low as 1.34 mM. With cAMP and cGMP as substrates, these two (121, and 122) molecules efficiently inhibited PDE3. This indicates that for the best anticancer action, suppression of both cAMP, as well as cGMP hydrolysis to boost intracellular concentrations of both signalling molecules may be necessary (Fig. 35). Utilizing cGMP as the substrate, compound 121 had an IC 50 of 10.9 mM and suppressed PDE3 (Table 8). Creating substrate-specic inhibitors for dual-functioning enzymes like PDE3 is a novel development that opens the way to modify the expression of one substrate instead of another, which may have implications for drug discovery in terms of safety and efficacy.
An adaption of the IMAP (Fluorescence polarization phosphodiesterase assay) was used to detect PDE activity.
3.9.2 Relationship between structure-propeties and anticancer activities. The cyclized pyridopyrimidone derivative 121 reduced the tumour cell proliferation, with an IC 50 of 1.34 mm (Fig. 36). With cAMP and cGMP as substrates, these three drugs efficiently inhibited PDE3. This indicated that the best strategy to treat cancer may be to limit both cAMP as well as cGMP hydrolysis to increase intracellular concentrations of both secondary messengers. The positive control milrinone's inability to cause apoptosis in the HT-29 cell line despite its dual inhibition of PDE3 and growth suggested that the other PDE and off proteins may contribute to the anticancer activity. Alkoxy and Bromo substituents at the ortho positions of the phenyl at positions 4 and 6, respectively, are believed to be responsible for the non-coplanarity between the two aryls and pyridone. The enforcement of bromophenyl non-coplanarity appears to be necessary for stacking with Phe991. Replacement of the oxygen atom of cyano-2-pyridones with an amino group may create the foundation for substrate-selective pharmacological regulation of this signicant class of therapeutic targets (Fig. 35). 121

Gene Kirsten rat sarcoma viral oncogene homolog inhibitors
The Gene Kirsten rat sarcoma viral oncogene homolog is known as KRAS. It belongs to the group of epidermal growth factor receptor (EGFR) kinase. This multi-component signaling system helps to regulate cell growth, division, survival, and death by relaying signals from the outside to the inside of the cell. An essential signal for cellular differentiation in normal cells is the interaction of the epidermal growth factor to its target, mainly on the cell membrane. There are two more signals in the process including tyrosine kinase enzymes as well as a protein encoded by the KRAS gene, instead of separately promoting cell proliferation, the route's components typically cooperate to control cell division and growth. KRAS mutations are frequently linked to resistance to targeted medicines and poor outcomes in cancer patients, yet aer more than three decades of research, no selective KRAS inhibitor has been authorized. However, in some tumors, EGFR becomes active even when EGF is not present, resulting in uncontrolled cell proliferation and division. EGFR or tyrosine kinase enzyme inhibitors are frequently used to treat these malignancies. However, an alteration in the KRAS gene found in a few of these malignancies leads to an abnormal K-Ras enzyme. The uncontrolled protein is constantly active and can promote cell growth despite the lack of signals from EGFR as well as other tyrosine kinase enzymes. Inhibitors of EGFR or tyrosine kinases will be ineffective in such tumours. [129][130][131] KRas is the most oen mutated oncogene among all types of human cancer. Aer the initial discovery of Ras oncogenes in 1982, In Ras-driven cancer, no approved medication specically targets Ras. 132 Ras proteins are chemical messengers that alternate between an active GTP-bound as well as an inactive GDP-bound state to control a number of cellular functions. Guanine nucleotide exchange factors (GEFs) enhance activation, and GTPase-activating proteins (GAPs) inactivates Ras protein by catalyzing GTP hydrolysis and are involved in these state changes. 133,134 Ras protein mutations result in anomalies in the switch mechanism, a signicant contributor to tumorigenesis. RAS mutations are present in three of the most lethal kinds of human malignancy, or around 25% of human tumors, including pancreatic cancer, and colon and lung cancer. KRas is the most frequently altered isoform of the Ras proteins (85%), followed by NRas (11%) and HRas (4%), with the most common mutations occurring at amino acid positions G12, G13, and Q61. 132 Mutations at positions 12 and 13, which cause a steric clash with the catalytic arginine nger donated by the GAPs, are thought to be oncogenic. In contrast, mutations at position 61 disrupt the coordination of a nucleophilic water molecule, resulting in an accumulation of active GTP-bound Ras proteins in cells. 135,136 The Shokat laboratory discovered the switch-II pocket in 2013, creating an opportunity for the creation of the KRasG 12C inhibitors, which is investigated in the trials. A library of 480 tethering compounds was screened for covalent binding to KRasG 12C using a disuldefragment-based screening method. 137 Structure-activity-relationship (SAR) investigations of 6H05, compound 6Ostrem, and KRAS G12C in the GDP state resulted in a co-crystal structure with KRAS G12C . Because crystallographic study revealed that the ligand does not attach to the nucleotidebinding pocket (S-IIP), it was given the switch-II pocket and placed near the activator binding switch-II. S-IIP is positioned between the core-sheet and switch-II, and when ligand binding occurs, it undergoes signicant conformational changes to form a separate pocket, whereas switch-I conformation remains intact from the GDP-bound state. 132 Many novel covalent allosteric KRAS G12C inhibitors have been created as a result of this pioneering work, including ARS1620 (124), 138 AMG510 (125), 139,140 and MRTX849 (126), 141,142 with AMG510 being the rst KRAS G12C inhibitor to reach clinical trials in August 2018 (Fig. 37). The ndings of a phase I trial using this medication were recently released, revealing partial responses in 50% of non-small cell lung cancer (NSCLC) patients and stable disease in the majority of colorectal or appendix cancer patients, all of whom carried the KRAS G12C mutation. 143 Xiao et al., 2021, designed new KRAS G12C inhibitors using a novel approach. Two sets of pyrido[2,3-d]pyrimidine-and pyridinyl N-atom-shied bicyclic pyrido [4,3-d]pyrimidinecontaining derivatives of the clinical KRAS G12C inhibitor AMG510 (125) were produced (Fig. 38). MIA PaCa-2 cells, clinical pancreatic cancerous cells with the KRAS G12C mutations, were used to test the tetracyclic derivatives; investigating potential off-target effects, the inhibitory activity on A549 cells carrying other KRAS mutant G12S has also been investigated. Although compound 128 demonstrated considerable efficacy, including an IC 50 value of 7.97 mM in MIA PaCa-2 cell line expressing KRAS G12C , the tetracyclic molecules still exhibited selectivity toward the G12C mutation of KRAS (Table 9). According to the docking study, the shadow connection between 128 in the front entry and an open hydrophobic pocket may be the main reason for its less efficacy. The piperazine derivative was less effective than the 3,8-bicyclo[3.2.1]octane linker in the parent skeleton. However, it was found that the piperizinyl linker, which joins the bond formation warhead towards the tetracyclic nucleus, had some biological efficacy. Compounds 127 and 128 have the most effective R 2 -substituents, such as methyl or uoro group. 143 The more robust character of (pyrido [4,3-d]pyrimidin) in comparison to 128 (pyrido[2,3-d]pyrimidin), explained by uorophenol group engaging a hydrophobic pocket and creating an H-bonds to Arg68. Additionally, the uorophenol molecule appeared to occupy the space by the tiny uoro group at the C-8 place (as in ARS1620). Only molecule 128 showed minor activity in the MIA PaCa-2 human cell expressing KRAS G12C , with IC 50 of 7.97 mM, whereas the tetracyclic molecules still showed speci-city for the G12C form of KRAS. According to structural docking study, the shadow contact of 127 at the front entry and an unoccupied hydrophobic pocket may be the primary cause of its weak potency. In biochemical and cellular experiments, bicyclic compound 128 was found to be substantial inhibitor. 143

Fibroblast growth factor receptors inhibitors
Several endothelial and tumour cells contain tyrosine kinases called FGFR (broblast growth factor receptors). They participate in tumour angiogenesis, tumour cell migration, survival, and proliferation. Many human malignancies have been connected to FGFR overexpression or aberrant activity control. As a result, reducing tumor cell proliferation, survival, and migration, as well as tumor angiogenesis by targeting FGFRs is   144 The activation of downstream transduction pathways occurs when the intracellular kinase domain of the FGFRs undergoes transautophosphorylation due to the binding of FGFs, which also causes the dimerization of FGFRs. 145,146 FGFRs contribute to crucial physiological activities, including cell migration, proliferation, differentiation, and survival, via activating downstream signalling pathways. 147-149 3.11.1 FGFR signaling pathways. FRS2, a prominent FGFR substrate, binds to the juxta membrane area of FGFR constitutively via its phospho-tyrosine binding domain (PTB), regardless of the kinase domain's activity or phosphorylation state. Several tyrosine residues in FRS2 are phosphorylated aer FGFR activation and serve as docking sites for the following molecules. 150 The RAS-MAPK-ERK signalling cascade is repeatedly activated by FRS2. RSA was chacterised by docking with different proteins, including SHP2 (SH2-containing tyrosine kinase phosphatase), GRB2 (growth factor receptor-bound protein 2), SOS (Son of Sevenless). The Src symmetry domain (SH2 domain) in SHP2 as well as GRB2 could bind directly phosphorylated tyrosine residues in GRB2 and FRS2. As a result, either directly or through the creation of the SHP2-GRB2-SOS complex, the GRB2-SOS complex is recruited to FRS2. 151,152 In the RAS-MAPK-ERK signalling pathway, the complex then initiates a phosphorylation cascade. ERK1/2 is translocated from the cytoplasm to the nucleus aer activation. It regulates the activity of various transcription factors to inuence cell proliferation, differentiation, and signal transduction, making it one of the most persuasive signalling molecules in this pathway. 153 When GRB2-related binding protein 1 (GAB1) binds to GRB2's SH3 domain, it can phosphorylate tyrosine itself and be dragged into the complex. Similarly, PI3K with an SH2 domain binds to the phosphorylated tyrosine residues of GRB2, activating the PI3K-AKT signalling cascade. AKT's downstream effector molecules include the well-known mTOR, which is involved in cell metabolism, transcription, and other processes (Fig. 39). 154 In addition to FGFR, PIP2 (phosphatidylinositol 4,5bisphosphate) is hydrolyzed by phospholipase C, which connects to a phosphorylated tyrosine in the kinase domain's Cterminus to produce the secondary mediators IP3 (inositol triphosphate) and DAG (diacyl glycerol). When IP3 binds to its receptor on the endoplasmic reticulum, Ca 2+ is released from intracellular reserves, increasing Ca 2+ concentration. 155 DAG activates the PKC signalling pathway when it is coordinated with Ca 2+ , which produces crosstalk with the RAS-MAPK pathway (due to the rivalry between GRB2 and PLCg) for FGFR binding. 156 Connolly et al. from the Parke-Davis laboratory were the rst to identify drugs tailored to target FGFR selectively. The authors used high-throughput screening approaches to nd FGFR inhibitors. This led to the discovery of pyrido[2,3-d]pyrimidine (129), which inhibited FGFR potently (IC 50 = 0.45 mM) in a biochemical assay; this indicates that the compound 129 binds at the ATP-binding site, and the molecule behaved in an ATP-competitive fashion. 157 3.11.2 Structure-activity relationship of FGFR inhibitors. SAR analysis revealed the creation of new analogs that were more potent, soluble, and bioavailable than the parent lead. Compound 129 was transformed into compound 131 [by introducing a {4-(diethylamino) butyl} amine substituent], which had a higher potency as well as availability with an IC 50 of 0.3 mM and reduced PDGF-stimulated vascular smooth muscle cell growth. A selective FGFr tyrosine kinase antagonist, 130, was also created by substituting 6-(2,6-dichlorophenyl) group of 129 with the 6-(3 ′ ,5 ′ -dimethoxyphenyl). If, the pyrido [2,3-d] pyrimidine nucleus has a N ′ -substituted alkylurea group on the seventh position of parent skeleton, excellent tyrosine kinase antagonists can be synthesiszed. 157 Compound 131 inhibited the FGFr tyrosine kinase with an IC 50 of 0.060 M, whereas the IC 50 s for the PDGFr, FGFr, EGFr, c-src, and InsR tyrosine kinases were all greater than 50 mM (Fig. 40 and 41). 157 4 Summary of anticancer activity of pyrido [2,3-

d]pyrimidine derivatives
The overall SAR for the anticancer activity of pyrido [2,3-d] pyrimidine derivatives is shown below in Fig. 42.

Future perspective
Many FDA approved drugs like palbociclib, pamapimod, and piritrexim, having the pyrido[2,3-d]scaffold in their structure and are used to treat different form of cancer by via inhibiting CDK4/6, p38, and DHFR respectively. AMG510 and R1478 are under phase 1 clinical trails against KRAS and p38 respectively. Dilmapimod (100), TAK-733 (101), and compound 102 have been withdrawn from clinical trials due to undesirable side effects. Therefore, there is need to design selective molecules to  combat resistance and problem of undesirable side effects. In this study, we have found many compounds showed excellent inhibition of cancer at the concentration of nM range. Researchers may design potent and selective molecules keeping in the consideration of stuructural requirements given in structure activity relationship study (Fig. 42).

Conclusion
Nitrogen containing heterocyclic compounds, especially pyrido [2,3-d]pyrimidines are a diverse class of chemicals having a number of biological properties. Tyrosine kinase, extracellular regulated protein kinases-ABL kinase, phosphatidylinositol-3 kinase, mammalian target of rapamycin, p38 mitogenactivated protein kinases, BCR-ABL, dihydrofolate reductase, Cyclin-dependent kinase, Phosphodiesterase, KRAS (the Gene Kirsten rat sarcoma viral oncogene homolog), and broblast growth factor receptors are the reported anticancer tagets of pyrido [2,3-d]pyrimidine. These derivatives have demonstrated signicant anticancer action upon modications at C-2, C-3, C-5, C-6, C-7, as well as C-8. Many researchers have explored its Structure activity relationship, as well as conformation and alignment parameters for ligand binding, employing computational modeling as well as docking investigations. The current study also emphasizes a variety of conventional, multicomponent, and microwave-assisted approaches for synthesizing pyrido [2,3-d]pyrimidine derivatives and will also help scientists nd highly effective, precise, and targeted pharmaceuticals.

Conflicts of interest
The authors declare no competing interests.